FOXM1 is critical for the fitness recovery of chromosomally unstable cells

Tumor progression and evolution are frequently associated with chromosomal instability (CIN). Tumor cells often express high levels of the mitotic checkpoint protein MAD2, leading to mitotic arrest and cell death. However, some tumor cells are capable of exiting mitosis and consequently increasing CIN. How cells escape the mitotic arrest induced by MAD2 and proliferate with CIN is not well understood. Here, we explored loss-of-function screens and drug sensitivity tests associated with MAD2 levels in aneuploid cells and identified that aneuploid cells with high MAD2 levels are more sensitive to FOXM1 depletion. Inhibition of FOXM1 promotes MAD2-mediated mitotic arrest and exacerbates CIN. Conversely, elevating FOXM1 expression in MAD2-overexpressing human cell lines reverts prolonged mitosis and rescues mitotic errors, cell death and proliferative disadvantages. Mechanistically, we found that FOXM1 facilitates mitotic exit by inhibiting the spindle assembly checkpoint (SAC) and the expression of Cyclin B. Notably, we observed that FOXM1 is upregulated upon aneuploid induction in cells with dysfunctional SAC and error-prone mitosis, and these cells are sensitive to FOXM1 knockdown, indicating a novel vulnerability of aneuploid cells.


INTRODUCTION
Chromosomal instability (CIN), defined as the continuous loss or gain of chromosomes is the result of increased levels of mitotic errors and aneuploidy [1]. Although CIN promotes tumor evolution, drug resistance and tumor heterogeneity, excessive CIN leads to cell death [2][3][4]. The mechanisms conferring cancer cells tolerance to high levels of CIN are not fully understood [5]. The identification of specific targets against these bypass mechanisms could provide therapeutic strategies against CIN tumors [6,7].
The spindle-assembly checkpoint (SAC) stalls the onset of anaphase until all kinetochores are properly bounded to microtubules during metaphase, preventing chromosome errors and CIN [8,9]. Overexpression of SAC proteins is common among human cancers and defective SAC functioning facilitates ongoing CIN [10,11]. Among these SAC proteins, MAD2 has been found to be upregulated in different types of cancer and its overexpression, in transgenic mouse models, induces mitotic arrest and increases the number of mitotic errors [12]. In addition, Mad2 overexpression in Kras-driven mouse breast tumors leads to increased somatic copy number alterations compared with Kras tumors [13]. However, how tumor cells overcome Mad2-induced mitotic arrest and tolerate Mad2-induced CIN is still unclear. Understanding the molecular mechanism behind could reveal novel therapeutic strategies for unstable MAD2-overexpressing cancers.
Microtubule (MT) poisons block mitosis by interfering with microtubule dynamics and therefore activating the SAC and have been widely applied in the treatment of solid cancers [14,15]. Intriguingly, clinical trials indicate that the cytotoxic effect of microtubule-targeting drugs might not only rely on the induced mitotic arrest but also in the excessive CIN generation [16].
One key mediator of anti-mitotic therapeutic response is the Forkhead Box M1 (FOXM1) transcription factor, which is involved in mitotic progression, spindle assembly and chromosome segregation [17][18][19]. FOXM1 preserves mitotic spindle formation and prevents mitotic catastrophe induced by the MT poison paclitaxel [20] while repression of FOXM1 increases paclitaxelinduced mitotic cell death through modulation of the apoptotic pathway [21]. FOXM1 also improved age-associated mitotic defects in elderly human dermal fibroblasts, leading to decreased aneuploid levels [22].
Here, we identify FOXM1 to be essential for the survival of tumor cells with MAD2 overexpression (OE) in mouse and human cell lines. FOXM1 overexpression facilitates mitotic exit and maintains chromosome segregation fidelity of MAD2overexpressing and nocodazole-treated cells by disrupting SAC signaling. Analysis of human tumors showed that high FOXM1 expression and increased aneuploidy were associated with poor prognosis, and cells with tetraploidization were more sensitive to depletion of FOXM1. Our results revealed that the upregulation of FOXM1 is a mechanism that allows cells to bypass mitotic arrest and tolerate CIN in MAD2-overexpressing cells.
Concentrations of nocodazole (Sigma, 487928) were 200 ng/ml, as indicated in figure legends. Tetraploid MCF7 and CAL51 were generated by cytokinesis inhibition using 0.75 μM dihydrocytochalasin B (DCB, inhibitor of actin polymerization, Sigma-Aldrich D1641) for 18 h overnight. Afterwards, cells were washed 3 times with PBS and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin for an additional 20 h.

Quantitative PCR
Frozen tissue from mice was ground with mortar and pestle on dry ice and RNA was purified using RNeasy Mini Kit (Qiagen

Statistical analysis
Prism9 was used for statistical analysis. Statistical analyses between two groups were carried out using unpaired t-test, between more than two F. Pan et al.
groups were performed with one-way analysis of variance, followed by Tukey's multiple comparisons test or two-way analysis of variance, followed by Sidak's multiple comparisons test. P-values were indicated in figure legends. P-value < 0.05 was considered as significant. Scatterplots were shown as mean and SEM. Points and connecting lines were shown as mean and error with SEM. Cell number and animal number were presented as n.

RESULTS
High levels of MAD2 confer aneuploid cells sensitivity to FOXM1 inhibition To understand how the genetic landscape in human tumors is altered by MAD2 expression, we performed independent analysis from data obtained from a human cancer cell line dataset (DepMap, n = 1389) and human breast tumors (GSE102484, n = 683; GSE54002, n = 417; GSE76275, n = 265). Cancer cell lines and GSE tumors were divided into sextiles according to MAD2 expression levels. Differential expression analysis between the top and bottom sextiles revealed 62 differentially expressed genes (62 DEG) that were frequently upregulated in all MAD2_high cancer cell lines and MAD2_high tumors (P < 0.01; log2 fold change > 1.5) ( Supplementary Fig. 1A).
Next, to understand how cells with high levels of MAD2 are able to tolerate CIN, we investigated the vulnerabilities of aneuploid MAD2_high cell lines. Cancer cell lines from DepMap were divided into quartiles based on their aneuploidy score (AS) [26]. Aneuploid cell lines (Top AS quartiles) were further distributed into sextiles according to their MAD2 levels. MAD2_high (Top sextile) and MAD2_low (Bottom sextile) groups were considered for further analysis. The cell lines in the low AS quartile were considered the near euploid group. Based on this stratification, we performed a comparison of genetic dependency between MAD2_high aneuploid (aneuploid with high levels of MAD2) and euploid cell lines (Fig. 1A). Analysis of CRISPR-Cas9 datasets revealed that 115 genes were essential in MAD2_high aneuploid cells (Fig. 1B, Table 1) [26]. Three genes (RACGAP1, ASPM, FOXM1) were present in both DEGs and CRISPR datasets ( Supplementary Fig. 1B). We then investigated RNAi (Achilles+DRIVE) datasets to look for genes whose depletion was more lethal to MAD2_high aneuploid cell lines than to euploid ones. We identified 39 differential dependencies of MAD2_high aneuploid cells (Fig. 1C, Table 1) and confirmed that FOXM1 was a common dependency for MAD2_high aneuploid cells in both RNAi and CRISPR datasets (Fig. 1D). Further analysis showed that MAD2_high aneuploid cells exhibited increased Relative dependency Statistical significance(-LOG 10 P) MAD2_high aneuploid Euploid Fig. 1 Identification of FOXM1 as a vulnerability of MAD2 high aneuploid cells. A Schematic of our comparison of genetic and chemical dependencies between MAD2 high and low cancer cell lines. Cell lines were assigned aneuploidy scores (AS), and the genetic and drug sensitivity landscapes were compared between the top and bottom AS quartiles. Cell lines in the top quartile of aneuploidy scores were divided into top and bottom sextiles according to their MAD2 expression (MAD2_high aneuploid vs near euploid cell lines and MAD2_low aneuploid vs near euploid cell lines). B Essential genes in MAD2_high aneuploid cells when compared to euploid cells, based on a CRISPR-Cas9 screen. The unique differential genetic dependencies in MAD2_high and euploid groups are shown in a volcano plot. The three genes present in both DEGs and CRISPR datasets are highlighted in red. C Essential genes for MAD2_high and euploid cell lines in RNAi datasets are shown as relative dependency. FOXM1 is highlighted in red. D The number of essential genes of MAD2_high aneuploid in RNAi and CRISPR datasets. E Comparison of aneuploidy scores and mRNA expression levels of MAD2 and FOXM1 between euploid and aneuploid cancer cell lines. ****P < 0.0001 and ***P = 0.0002; One-way ANOVA.      FOXM1 mRNA levels compared to euploid cells or MAD2_low aneuploid ones (Fig. 1E). FOXM1 controls cell cycle-related gene expression and regulates chromosome stability [27,28]. To understand if the requirement of FOXM1 is associated with aneuploidy or with MAD2 levels, we examined essential genes of MAD2_low aneuploid cells as well as those in MAD2_high euploid cells. In both cases, FOXM1 was not found in either CRISPR or RNAi datasets (Table 2). Moreover, CRISPR and RNAi datasets analyses indicated that MAD2_high euploid cells are not sensitive to FOXM1 inhibition ( Supplementary Fig. 1D). Since FOXM1 mRNA expression is similar in MAD2_high euploid and aneuploid cells ( Supplementary Fig. 1C), we hypothesized that FOXM1 essentiality might be related to MAD2 expression and aneuploidy status.
To further confirm if FOXM1 is crucial in MAD2_high aneuploid cells, we examined two different drug screening datasets between MAD2_high aneuploid cell lines and euploid cell lines (Fig. 1A). Drug sensitivity data (AUC) suggested that MAD2_high aneuploid cells were more sensitive than euploid cells to the FOXM1 inhibitor Thiostrepton (Supplementary Fig. 1E). Moreover, FOXM1 has been described to be a target for the proteasome inhibitors Bortezomib, Delanzomib and Oprozomib [29,30]. Analysis of PRISM drug dose-level dataset indicated that MAD2_high aneuploid cells were more sensitive to proteasome inhibitors than euploid cells ( Supplementary Fig. 1F). Altogether, these results suggest that aneuploid cells with high MAD2 levels are more vulnerable to FOXM1 inhibition.
FOXM1 inhibition decreases mitotic fidelity in short-term MAD2 overexpressing cells Dysregulation of FOXM1 disrupts mitosis and increases CIN [31,32]. To study the impact of FOXM1 inhibition on mitosis in MAD2-overexpressing cells (MAD2 OE), we generated doxycycline (Dox) inducible human MAD2-expressing breast cancer cell lines (MCF7, CAL51, MDA-MB-231, and MCF10A). MAD2 overexpression resulted in increased FOXM1 levels after 6 days ( Fig. 2A and Supplementary Fig. 2A and 2B) and reduced cell viability in all cell lines over time ( Fig. 2B and Supplementary Fig. 2C). We then used siRNAs against FOXM1 in these cell lines, infected with an empty vector or with the Dox inducible MAD2 vector and monitored the consequences of FOXM1-downregulation (FOXM1 KD) 6 days after induction. Cell viability was significantly reduced in MAD2 OE cells after FOXM1 KD compared to the inhibition of FOXM1 alone ( Fig. 2C and Supplementary Fig. 2D).
We then selected two of these cell lines, one unstable breast cancer cell line, MCF7 and one diploid cell line, CAL51, and performed live cell imaging to study if mitotic defects or mitotic timing were contributing to the reduced viability. Time-lapse microscopy revealed that FOXM1 KD prolonged mitotic duration in MAD2 expressing MCF7 and CAL51 cells but not in the ones infected with an empty vector ( Fig. 2D and F). In addition, we found a significant increase in the number of mitotic errors when FOXM1 was downregulated in both cell lines (Fig. 2E and G and Supplementary Fig. 2E, F). Strikingly, MAD2-expressing cell lines had the highest rates of mitotic errors after FOXM1 depletion, suggesting that FOXM1 depletion not only affected mitotic duration but also increased the incidence of lagging chromosomes and other mitotic errors when MAD2 was overexpressed.
We next generated mouse embryonic fibroblasts (MEFs) from KH2-Mad2/Rosa26-rtTA Dox inducible transgenic mice [13]. Induction of MAD2 after 30 h on Dox led to higher FOXM1 protein levels (Fig. 2H). We previously published that overexpression of MAD2 causes mitotic delay and CIN in these cells [12]. Inhibition of FOXM1 had no significant effect on mitotic duration in wild-type MEFs while it induced a mild mitotic delay in MAD2-overexpressing cells (Fig. 2I). In addition, we observed increased number of mitotic errors upon MAD2 OE or FOXM1 KD in cells that were further increased in the combination of both. Interestingly, an increase in cell death and mitotic errors was observed in MAD2 OE cells after FOXM1 KD (Fig. 2J). These results indicate a causal link between MAD2 induced CIN and FOXM1 dependency. Altogether, our results show that whereas normal cells tolerate the downregulation of FOXM1, it results in prolonged mitosis and increased CIN levels in MAD2 OE cells.  Finally, we used mammary epithelial (EP) cells from our TetO-HA-Mad2/MMTV-rtTA doxycycline-inducible transgenic mice (M) where short-term MAD2 overexpression has been shown to induce mitotic arrest and cell death [13]. Consistent with our observations in MAD2 OE MEFs, we observed high FOXM1 protein levels after MAD2 induction in EP cells (Fig. 2K) and immunofluorescence staining confirmed that tipically cells with HA-MAD2 overexpression showed FOXM1 upregulation (Supplementary Fig. 2G). We then measured the effect of FOXM1 downregulation using the RCM-1 inhibitor on 3D cultures grown from the EP of M mice [33]. FOXM1 is required for the fitness of high MAD2 unstable cells MAD2 overexpression in Kras-induced breast tumors leads to the formation of chromosomally unstable tumors that retained MAD2 expression [13], suggesting that these MAD2 tumor cells adapt over time to high MAD2 levels. To investigate, whether Foxm1 can play a role in this adaptation process, we asked if long-term induction of MAD2 also resulted in increased FOXM1 levels. We harvested breast tumor cells from TetO-Kras/MMTV-rtTA transgenic mice (K) and TetO-Kras/TetO-Mad2/MMTV-rtTA mice (KM) and observed higher Foxm1 mRNA and protein levels in KM compared to K tumor cells (Fig. 3A, B). To explore the transcription profiling of tumor cells, RNA sequencing data from K and KM tumors was analyzed [24]. Gene set enrichment analysis (GSEA) revealed downregulation in pathways related to APC/C:CDH1 function, chromosomal segregation and antiapoptotic signaling in KM tumors when compared to K tumors ( Supplementary Fig. 3A). These results suggest that long-term MAD2 overexpression might lead to a dysfunctional regulation of mitosis and apoptosis in mouse breast tumor cells. To understand the consequences of FOXM1 inhibition after long-term MAD2 expression, we used siRNA to knockdown Foxm1 in K and KM tumor cells. siFoxm1 for 6 days in KM cells resulted in a significantly reduced cell viability (Fig. 3C), the accumulation of G2/M cells (Fig. 3D) and increased TUNEL-positive cells (Fig. 3E) when compared to non-treated ones, while inhibition of Foxm1 in K cells had no effect. Moreover, treatment with the RCM-1 inhibitor or siFoxm1 led to increased levels of Cleaved Caspase-3 and Gamma-H2AX (γ-H2AX) in KM cells (Fig. 3F). Although total inhibition of FOXM1 was not achieved in KM cells, we still observed that KM unstable cells are significantly more vulnerable than K cells to the downregulation of FOXM1. Finally, we overexpressed MAD2 in MCF7 cells for a long time (20 days) and analyzed the effect of inhibiting FOXM1. siFOXM1 led to severe mitotic arrest in MAD2 OE cells ( Supplementary Fig.  3B), which further suggests an essential role of FOXM1 in mitotic segregation. We also noticed higher γ-H2AX in both parental and MAD2 OE MCF7 cell lines after FOXM1 inhibition, indicating a role of FOXM1 in regulating DNA damage [34]. Increased C-caspase3 levels in MAD2 OE cells with siFOXM1 illustrated that MAD2expressing cells still rely on FOXM1 for mitotic exit and survival ( Supplementary Fig. 3C). Collectively, these data suggest that FOXM1 is critical for the viability of CIN cells induced by MAD2 overexpression.
FOXM1 overexpression preserves mitotic fidelity in MAD2 overexpressing human breast cancer cell lines To test whether FOXM1 plays a role in MAD2 tolerance in human cell, we infected MAD2-expressing human breast cancer cell lines with either an empty vector (EV) or with doxycyclineinducible human FOXM1 lentiviral vector (expressing FOXM1b or FOXM1c isoforms). Overexpression of FOXM1 in these cell lines showed similar viability compared to cells infected with the EV six days after induction. However, overexpression of FOXM1 significantly decreased the lethality of MAD2-expressing cells (Fig. 4A) and decreased the percentage of TUNEL-positive MCF7 cells (Supplemental Fig. 4A), suggesting that high FOXM1 levels can rescue MAD2-induced defective cell fitness. Since FOXM1 regulates cell cycle progression as well as chromosome segregation [35], we next sought to understand whether FOXM1 upregulation interferes with MAD2-mediated mitotic arrest and CIN in these cell lines. We performed live-cell imaging of MCF7 and CAL51 cells with or without MAD2 OE and FOXM1 OE. The overexpression of FOXM1 alone did not affect mitotic duration or mitotic errors when compared to EV cells. However, cells overexpressing MAD2 showed a significant decrease in mitotic time and reduced mitotic errors when FOXM1 was overexpressed at the same time (Fig. 4B, C). These results indicate that upregulation of FOXM1 facilitates mitotic exit in MAD2-expressing cells, allowing proper chromosome segregation and consequently, reducing MAD2-induced CIN.
MAD2 overexpression suppresses Aurora B activity, resulting in hyperstable microtubule-kinetochore attachments [36]. We observed that AURORA B expression was suppressed during mitosis in MAD2 OE cells, whereas the levels were maintained when FOXM1 and MAD2 were overexpressed in MCF7 cells Fig. 2 FOXM1 inhibition is detrimental to cell fidelity of Mad2 overexpressing cells. A Western blots of FOXM1 and MAD2 in human breast cell lines infected with an empty vector (EV) or a Dox-inducible MAD2 expressing vector (MAD2 OE) after dox administration for 6 days. ACTIN was used as a loading control. Three biological replicates were analyzed. B Cell viability of each human cell line after dox treatment for 3 or 6 days. Each dox-treated cell line was normalized to the untreated one. MCF7: ***P < 0.0005; MDA-MB-231: ***P < 0.0001; CAL51: ****P < 0.0001, ***P = 0.0004, **P = 0.0027; MCF10A: **P < 0.008, *P = 0.0359, One-way ANOVA. Each dot is a biological replicate. C Cell viability of human cell lines after MAD2 overexpression and FOXM1 knockdown by siRNA for 6 days. Values of each cell line were normalized to those of each EV group. Each dot is a biological replicate. MCF7: *P = 0.0228; CAL51: *P = 0.0124; Two-way ANOVA. D Mitotic duration of MCF7 cells with or without MAD2 overexpression and siFOXM1 after 3 days. *P = 0.01, ****P < 00001; Two-way ANOVA. (EV, 100 cells; MAD2 OE, 92 cells;  FOXM1 KD, 95 cells; MAD2 OE/ ( Supplementary Fig. 4B). Thus, this result suggests that FOXM1 regulates AURORA B allowing MAD2-overexpressing cells to exit mitosis after the induced cell cycle arrest.
To test if FOXM1 overexpression can rescue the detrimental effects of MAD2 OE in vivo, MCF7 cells with empty vector, MAD2 OE or MAD2 together with FOXM1 OE were injected as xenografts in nude mice (Fig. 4D). In line with our in vitro results, persistent high MAD2 levels resulted in decreased fitness and a delayed tumor onset while the combined overexpression of MAD2 and FOXM1 reverted this effect (Fig. 4E). Ten weeks after injection, 2 out of 5 animals injected with MAD2 OE cells developed tumors while the additional overexpression of FOXM1 allowed for 4 out of 5 animals to grow tumors (Fig. 4E). However, FOXM1 did not affect tumor growth at early (day 40) or late (day 56) time points throughout tumor progression (Fig. 4F). Thus, FOXM1 overexpression was not sufficient to accelerate the growth of MAD2 tumors, but it contributed to the tolerance of MAD2-induced detrimental effects on tumor cells.
To corroborate whether FOXM1 endows cells with tolerance to aneuploidy and CIN during chronic MAD2 OE, we analyzed the outcome of cell division in human breast cell lines 20 days after MAD2 induction. MCF7 cells after a long expression of MAD2 continue to present prolonged arrest in mitosis. While long-term MAD2 upregulation resulted in an average mitotic time of 2.67 h, further FOXM1 overexpression was not able to shorten this time (average 2.23 h) (Supplementary Fig. 4C). We next compared CIN levels between MAD2 OE and MAD2/FOXM1 OE by monitoring mitotic errors. A lower percentage of mitotic errors was observed in MAD2 OE cells with constitutive overexpression of FOXM1 indicating a protective role of FOXM1 against CIN during long-term MAD2 OE. (Supplementary Fig. 4D, E). Interestingly, once cells adapted to high MAD2 levels, no cell death was observed in either MAD2 OE cells alone or in the combination with FOXM1 upregulation and the spectrum of mitotic errors was different from the ones seen after a short exposure to MAD2. Elevated FOXM1 and aneuploidy are associated with poor prognosis in BRCA patients Previous data indicated that upregulation of FOXM1 facilitates mitotic exit of MAD2-overexpressing cells in the presence of CIN. We reasoned that aneuploid cancers might also require FOXM1 to compensate for deleterious CIN and hypothesized that cancers that allow propagation of CIN could be associated with a worse survival rate. These results were validated in human breast cell lines and mouse breast tumors, however, to further test this hypothesis, we analyzed TCGA data of BRCA patients (n = 1064) and divided them into two groups (FOXM1_low and FOXM1_high) based on FOXM1 mRNA levels. We then calculated the aneuploidy levels of these samples by determining the mean absolute changes in the copy number segment. Aneuploidy was considered as a deviation from 0, which represents euploidy. FOXM1_low samples showed lower copy number alteration (SCNA) counts while FOXM1_high samples had higher SCNA counts (Fig. 5A). However, no difference in overall survival was observed between these two groups (Fig. 5B). To assess the survival of patients with different aneuploidy levels related to FOXM1 expression, we divided patients into two groups based on  SCNA counts (SCNA_low and SCNA_high). Patients with higher SCNA counts presented poor prognoses in FOXM1_high cancers, whereas changes in SCNA had no effect on the prognosis of FOXM1_low cancers (Fig. 5C). Patients with high FOXM1 expression and high SCNA had the worse survival compared to the other groups (Fig. 5D). In summary, these data suggest that survival of BRCA patients is not directly associated with FOXM1 levels. Instead, their outcomes are associated with the upregulation of FOXM1 and increased genomic alterations in the cancers. Thus, FOXM1 might facilitate tolerance of high aneuploid tumors and provide advantages for aneuploid cancer development.

FOXM1 facilitates mitotic exit after nocodazole-induced arrest
We speculated that increased FOXM1 levels antagonize mitotic checkpoint signaling and release cells from mitotic arrest. To test this hypothesis, we forced mitotic checkpoint activation by treating cells with the spindle poison nocodazole (Noco). A high dose of Noco treatment blocked cells in mitosis and in line with our previous results, FOXM1 overexpression released these cells from prolonged mitosis (Fig. 6A). In addition, high levels of FOXM1 partially rescued the mitotic slippage induced after Noco treatment (75% of mitotic slippage in Noco treated cells vs 68% in Noco/FOXM1overexpression), as well as cell death in mitosis (11% vs 2%) (Fig. 6B). SAC effectors including MAD2 and CDC20 are all involved in chromosome segregation [37]. Cyclin B is known to block mitotic exit regardless of SAC activity and is degraded by the CDC20-APC/C complex during mitosis [35]. We confirmed MAD2 and Cyclin B protein levels to be downregulated in Noco-treated FOXM1 OE cells compared to control cells after Noco treatment (Fig. 6C). This suggests that FOXM1 OE compromises SAC signaling and reduces Cyclin B in MCF7 cells, leading to chromosome segregation and exit from mitosis. Next, to test whether FOXM1 is responsible for allowing aneuploid cells to exit mitosis, we challenged CAL51 cells with Reversine, an MPS1 inhibitor that abrogates SAC signaling and promotes premature exit from mitosis [38]. While Reversine treatment in control cells or FOXM1 KD cells did not alter mitotic duration, mitotic arrest induced by MAD2 or Noco-was reverted after Reversine treatment in CAL51 cells (Fig. 6D). Importantly, Reversine was not able to rescue the prolonged mitosis in CAL51 cells when siFOXM1 was applied (Fig. 6D). These data suggest that FOXM1 is crucial to restoring mitotic exit in aneuploid cells with a hyperactivated SAC.
To further verify if FOXM1 is essential for aneuploid cells, we attempted to generate aneuploid cells by treating breast cancer cells with dihydrocytochalasin B (DCB), to block cytokinesis and induce tetraploidy [39]. Consistent with the previous reports [40,41], an increase of mitotic errors was observed after DCB treatment, suggesting that cells became chromosomally unstable (Fig. 6E, F). MCF7 and CAL51 cell lines exhibited increased FOXM1 protein levels after DCB treatment. In addition, CAL51 cells treated with DCB also had higher MAD2 levels compared to non-treated cells (Fig. 6G). We next inhibited FOXM1 in these unstable cells. Long-term inhibition of FOXM1 impaired proliferation, especially in the DCB-treated cells (Fig. 6H) suggesting that chromosomally unstable cells have increased sensitivity to FOXM1 inhibition.

DISCUSSION
In our current study, we report that depletion of Foxm1 in various cells that overexpress MAD2 (including human breast tumor cells, MEFs, normal mouse mammary epithelial cells and mouse tumor cells), leads to an extension of mitosis and an increase in mitotic errors. Tumor cells with high levels of MAD2 can adapt over time by accumulating FOXM1, which renders them vulnerable to FOXM1 inhibition. Our investigation has revealed that FOXM1 upregulation facilitates chromosome segregation and mitotic exit in human cell lines with high MAD2 levels by suppressing SAC signaling. As a result, these cells are rescued from their defective proliferation. Additionally, we have found that FOXM1 can reverse mitotic arrest and correct mitotic errors induced by other mechanisms such as nocodazole or cytokinesis failure.
Tumors can overcome the mitotic checkpoint induced by Mad2 overexpression and become aneuploid [12,13]. The increasing number of mitotic errors suggest that cells force mitotic exit and regain cell fitness despite acquiring CIN. Interestingly, high levels of CIN can actually be tumor-suppressive, making high CIN tumors a potential therapeutic opportunity to halt CIN accumulation in unstable cells [5].
MAD2 overexpression in cells is detrimental, but the mechanisms by which cells overcome this effect are not well understood. By analyzing the genetic dependencies in high MAD2 aneuploid cell lines we found that FOXM1 is an essential gene in these cells. FOXM1 can activate various cell cycle-related proteins and is closely associated with CIN, suggesting that FOXM1 might be relevant to tolerate chromosomal instability. Our findings support the idea that high MAD2 CIN cells might require FOXM1 for maintaining proliferative fitness. However, once MAD2 is overexpressed for an extended period of time, chromosome errors persist, and the number of polyploid cells increases, suggesting that cells can escape the mitotic arrest via slippage. Although multiple mechanisms can allow cells to escape from mitotic arrest [15,42], we found that the level of SAC proteins decreases in MAD2 overexpressing cells with FOXM1 upregulation, providing insights into the role of FOXM1 in the maintenance of chromosomal stability.
Similarly, inhibition of FOXM1 increases the levels of these SAC proteins, strengthening the checkpoint and inducing mitotic arrest and cell death. We conclude that FOXM1 is required for proliferative fitness in high MAD2 cells with CIN. Nevertheless, FOXM1 prevents mitotic cell death in cells with paclitaxel treatment [21], our results cannot exclude the possibility of cell death prevention of FOXM1during MAD2 overexpression.
FOXM1 insufficiency causes centrosome abnormalities and disrupts the spindle formation [43,44]. Thus, FOXM1 is a valuable target to increase mitotic stress in CIN cells. Consistent with this, we find that short-term FOXM1 inhibition is insufficient to arrest cells in mitosis but leads to lagging chromosomes and chromosome misalignments in human cell lines. As mitotic errors accumulate, several cellular outcomes are expected. First, the mitotic checkpoint is activated leading to mitotic arrest [11]. Second, DNA damage and replication stress block cell cycle entry, leading to quiescence and senescence [45]. Finally, overwhelmingly high CIN levels activate apoptotic pathways and induce cell death [46]. In agreement, our results demonstrate that high levels of MAD2 confer aneuploid cell lines sensitivity to FOXM1 inhibition, reducing their proliferative ability as long-term FOXM1 inhibition induces high levels of mitotic aberrations. Thus, FOXM1 represents a critical target to tumor cells exhibiting CIN or aneuploidy.

DATA AVAILABILITY
All data generated and analyzed during the study are available from the corresponding author upon reasonable request.